The goal of studies proposed in this application is to elucidate the molecular mechanisms that regulate expression of the adaptor protein HSH2 in the B cell lineage and to structurally and functionally characterize low molecular weight (LMW) species of HSH2 expressed uniquely in germinal center B cells. Proper function of the humoral immune response is dependent on the process of class switch recombination, which involves recombination of variable gene elements on the immunoglobulin heavy chain with distal constant region gene elements to generate class switched antibodies that exhibit the same antigenic specificity, but have the ability to perform distinct effector functions required for mediating an optimal immune response against pathogens. Expression of the adaptor protein HSH2 is tightly regulated in B cell subpopulations in the periphery. High-level full length HSH2 expression is observed in innate like B cells (marginal zone and B1a/b B cells) that do not undergo class switching. Conversely, low-level full length HSH2 expression is observed in follicular and germinal center B cells that ultimately generate class-switched antibody secreting cells. Analysis of mouse models expressing high versus low levels of HSH2 have demonstrated that the expression of HSH2 inversely correlates with the production of class-switched antibody secreting cells and class-switched antibody in response to challenge with model antigens. The scientific premise of the proposed studies is that regulation of HSH2 expression plays a critical role in controlling the production of class-switched ASCs and Abs, and that delineation of the molecular mechanisms that regulate HSH2 expression are highly significant with respect to understanding how the humoral immune response is regulated. Two specific aims are proposed in this application. Because preliminary studies have shown that HSH2 expression is regulated via a post-translational mechanism mediating its turnover, experiments in Specific Aim 1 will be performed to analyze the B cell degradome, transcriptome and proteome to identify the protease responsible for rapid degradation of HSH2 in B cells. These studies have the potential to identify a novel post-translational regulatory process that plays a critical role in controlling the production of class-switched antibodies. Specific Aim 2 will focus on structural and functional characterization of LMW species of HSH2 that are uniquely expressed in germinal center B cells and that may play an important role in regulating the germinal center response leading to production of class-switched antibodies. Mass spectrometry will be used to characterize LMW species of HSH2, which will then be expressed via retroviral transduction of stem cells and adoptive transfer experiments to assess their function during the germinal center response following challenge with T-dependent antigen. The proposed studies are highly significant with respect to the development of strategies to manipulate the humoral response to enhance vaccine efficacy, as well as the humoral response against microbial pathogens. !